This revision of NIH P41RR001614 requests support to expand our development of a new class of chemical cross-linking reagents to address new Biodriver needs for integrated, robust methodology that will yield distance constraints for modeling of the architecture of much larger protein complexes and machines. The new Biodriver Project is entitled, Architecture of RNA polymerase II Transcription Initiation Complexes, the Principal Investigator is Roger Kornberg of Stanford University. Our plan is to build upon our recent successful TR&D demonstration that reductive amination of aryldialdehydes with e-aminofunctions on lysine side chains is virtually ideally suited to sequence and structural characterization by electron transfer dissociation (ETD) mass spectrometry using the Velos Orbitrap platform. Virtually complete sequences are obtained from ETD spectra for BOTH peptides in the cross-linked species. Also gas phase enrichment of peptide cross-links Is achieved by selection of precursor charge state (eliminate states below +4) for ETD spectral recording, this is possible because reductive amination generates two new secondary amines that are protonated in electrospray ionization. Kornberg's laboratory has highly purified complexes that range in molecular size from some 30 proteins to more than 100. Taking advantage of this challenging array of related complexes, we will be able to optimize and exploit the major inherent advantages of our new integrated experimental strategy (reagents, optimal conditions for cross-linking and subsequent digestion, ETD MS and new software and bioinformatics tools to facilitate and accelerate progress). Once a robust protocol is optimized using our new integrated strategy, this capability will be exported to the biomedical research community. Almost untold numbers of such protein complexes exist that carry out a host of processes and functions in human and other cells. For example, we have just reported the characterization of the HIV-human host protein interactome landscape that provides evidence of almost 500 complexes (Jaeger et al., 2012 Nature, 481, 365-370,doi:10.1038/nature10719).